Transferrin receptor protein of Moraxella

ABSTRACT

An isolated and purified non-denatured transferrin receptor protein of a Moraxella strain, particularly  M. catarrhalis , has an apparent molecular mass of about 80 to about 90 kDa, as determined by SDS-PAGE. The transferrin receptor protein or a fragment analog thereof is useful in diagnostic applications and immunogenic compositions, particularly for in vivo administration to a host to confer protection against disease caused by a strain of Moraxella.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with transferrin receptor protein from Moraxella,methods of production, and uses thereof.

BACKGROUND OF THE INVENTION

Otitis media is the most common illness of early childhood withapproximately 80% of all children suffering at least one bout of otitismedia before the age of three (ref. 1—Throughout this application,various references are referred to in parenthesis to more fully describethe state of the art to which this invention pertains. Fullbibliographic information for each citation is found at the end of thespecification, immediately preceding the claims. The disclosures ofthese references are hereby incorporated by reference into the presentdisclosure). Chronic otitis media can lead to hearing, speech andcognitive impairment in children. It is caused by bacterial infectionwith Streptococcus pneumoniae (approximately 50%), non-typableHaemophilus influenzae (approximately 30%) and Moraxella (Branhamella)catarrhalis (approximately 20%). In the United States alone, treatmentof otitis media costs between one and two billion dollars per year forantibiotics and surgical procedures, such as tonsillectomies,adenoidectomies and insertion of tympanostomy tubes. Because otitismedia occurs at a time in life when language skills are developing at arapid pace, developmental disabilities specifically related to learningand auditory perception have been documented in youngsters with frequentotitis media.

M. catarrhalis mainly colonizes the respiratory tract and ispredominantly a mucosal pathogen. Studies using cultures of middle earfluid obtained by tympanocentesis have shown that M. catarrhalis causesapproximately 20% of cases of otitis media (ref. 2).

Long regarded as an opportunistic pathogen, B. catarrhalis is nowrecognized to cause a variety of potentially debilitating human diseasesas a result of localized or, more rarely, systemic infection.

M. catarrhalis is an important cause of lower respiratory tractinfections in adults, particularly in the setting of chronic bronchitisand emphysema (refs. 3, 4, 5, 6, 7, 8 and 9). M. catarrhalis also causessinusitis in children and adults (refs. 10. 11, 12, 13 and 14) andoccasionally causes invasive disease (refs. 15, 16, 17, 18, 19 and 20).In the hospital setting, M. catarrhalis has been suspected in outbreaksof nosocomial infection (ref. 21).

The incidence of otitis media caused by M. catarrhalis is increasing. Asways of preventing otitis media caused by pneumococcus and non-typeableH. influenzae are developed, the relative importance of M. catarrhalisas a cause of otitis media can be expected to further increase. Alsoantibiotic resistance is becoming common in clinical isolates of M.catarrhalis (ref 22). Thus, prior to 1970 no β-lactamase producing M.catarrhalis strains had been reported, but since the mid seventies,β-lactamase expressing strains have been detected with ever increasingfrequency among isolates. Recent surveys suggest that 75% of clinicalisolates produce β-lactamase.

Iron-restriction is a general host defence mechanism against microbialpathogens, however, it is not necessarily growth rate-limiting for allpathogens (ref. 23). A number of bacterial species including Neisseriameningitidis (ref. 24), N. gonorrhoeae (ref. 25) and Haemophilusinfluenzae (ref. 26), expresses two outer membrane proteins whichspecifically bind human transferrin (refs. 27, 28). The expression ofthese proteins is regulated by the amount of iron in the growthenvironment. Unlike the receptors of other bacterial species, the M.catarrhalis receptors have a preferred affinity for iron-laden (i.e.,ferri-) transferrin (ref. 29). The two M. catarrhalis transferrinreceptors (TfR) have molecular masses of 115 kDa (TfR1) and about 80 to90 kDA (TfR2) (ref. 27).

The outer membrane protein OMP B2 (refs. 30, 31) has a molecular masssimilar to that of TfR2, and it has been reported that the expression ofOMP B2 is iron-regulated (ref. 32).

Yu and Schryvers (ref. 29) describe a method for purification of thetransferrin receptor proteins TfR1 and TfR2 from M. catarrhalis byselective elution from a transferrin-sepharose affinity column using thedenaturing agent guanidine HCl.

In this method of the preparative isolation of receptor proteins, asuspension of iron-deficient crude M. catarrhalis membranes wassolubilized by the addition of EDTA to 20 mM and Sarkosyl to 0.75% andthen the mixture was centrifuged for 15 minutes at 20,000×g to removedebris. Twenty ml of Fe₂hTf-Sepharose (i.e. an iron-saturated form ofhuman transferrin) was added to the supernatant and then incubated withmixing at room temperature for 45 minutes. The mixture was applied to acolumn and after removing the binding solution, the resin was washedwith an additional 250 ml of 50 mM Tris-HCl, 1 M NaCl, 20 mM EDTA, 0.75%Sarkosyl, pH 8 buffer containing 250 mM guanidine HCl. The TfR2 proteinwas eluted using a buffer (lacking Sarkosyl) containing 1.5 Mguanidine-HCl and subsequently the TfR1 protein was eluted by theapplication of a buffer containing 4 M guanidine-HCl. The fractions weredialysed against 50 mM Tris HCl, pH 8 over a 24 hour period.

In a further modification of this procedure, apohTf-Sepharose was mixedwith the solubilized membrane preparation in order to bind TfR1 and thenthe treated solubilized membrane preparation was exposed toFe₂hTf-Sepharose to bind the remaining TfR2. The affinity resins weresubsequently washed and the receptor proteins eluted as described above

M. catarrhalis infection may lead to serious disease. It would beadvantageous to provide non-denatured transferrin receptor protein fromM. catarrhalis for use as antigens in immunogenic preparations includingvaccines, carriers for other antigens and immunogens and the generationof diagnostic reagents.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision of purified andisolated transferrin receptor protein of Moraxella catarrhalis and otherMoraxella strains, having an apparent molecular mass of about 80 to 90kDa.

In accordance with one aspect of the invention, there is provided anisolated and purified, non-denatured transferrin receptor protein of aMoraxella strain having an apparent molecular mass of about 80 to about90 kDa, as determined by sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE), or a fragment or an analog thereof (TfR2).The transferrin receptor protein may be substantially in its nativeconformation (so as to have substantially retained the characteristicimmunogenicity of the transferrin receptor protein in the Moraxellastrain) and may be isolated from a M. catarrhalis strain, such as fromM. catarrhalis 4223, 5191 or 135. Such isolated and purified transferrinreceptor protein of apparent molecular mass of about 80 to about 90 kDais substantially free from non-80 to 90 kDa Moraxella proteins,particularly the OMP B2 protein and the transferrin receptor proteinhaving an apparent molecular mass of about 105 kDa of the Moraxellastrains, phospholipids and lipopolysaccharide of Moraxella. The about 80to about 90 kDa transferrin receptor protein is at least about 70 wt %pure, preferably at least about 90 wt % pure, and may be in the form ofan aqueous solution thereof.

The present invention also provides an immunogenic compositioncomprising an immunoeffective amount of an active component, which maybe the transferrin receptor protein or fragment or analog thereof,provided herein along with a pharmaceutically acceptable carriertherefor. The immunogenic composition may be formulated as a vaccine forin vivo administration to a host to confer protection against diseasescaused by a strain of Moraxella, particularly M. catarrhalis. Theimmunogenic composition may be formulated as a microparticle capsule,ISCOM or liposome preparation. The immunogenic composition may be usedin combination with a targeting molecule for delivery to specific cellsof the immune system or to mucosal surfaces. Some targeting moleculesinclude strain B12 and fragments of bacterial toxins, as described in WO92/17167 (Biotech Australia Pty. Ltd.), and monoclonal antibodies, asdescribed in U.S. Pat. No. 5,194,254 (Barber et al). The immunogeniccompositions of the invention (including vaccines) may further compriseat least one other immunogenic or immunostimulating material and theimmunostimulating material may be at least one adjuvant. Suitableadjuvants for use in the present invention include, (but are not limitedto) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivativesand components thereof, calcium phosphate, calcium hydroxide, zinchydroxide, a glycolipid analog, an octadecyl ester of an amino acid, amuramyl dipeptide, polyphosphazene and a lipoprotein. Advantageouscombinations of adjuvants are described in copending U.S. patentapplication Ser. No. 08/261,194 filed Jun. 16, 1994, assigned to theassignee hereof and the disclosure of which is incorporated herein byreference. The invention further includes an antibody specific for thetransferrin receptor protein provided herein producible by immunizing ahost with an immunogenic composition as provided herein.

The immunogenic compositions provided herein may be formulated tocomprise at least one additional immunogen, which may comprise or bederived from a paramyxovirus, Chlamydia, polio, hepatitis B, diphtheriatoxoid, tetanus toxoid, influenza, haemophilus, pertussis, pneumococcus,mycobacteria and hepatitis A.

In a further aspect of the invention, there is provided a method ofgenerating an immune response in a host, comprising administeringthereto an immuno-effective amount of the immunogenic composition asprovided herein. The immune response may be a humoral or a cell-mediatedimmune response. Hosts in which protection against disease may beconferred include primates including humans.

The present invention provides, in an additional aspect thereof, amethod of producing a vaccine comprising administering the immunogeniccomposition provided herein to a test host to determine an amount and afrequency of administration of the transferrin receptor protein toconfer protection against disease caused by a strain of Moraxella andformulating the transferrin receptor protein in a form suitable foradministration to a treated host in accordance with said determinedamount and frequency of administration. The treated host may be a human.

A further aspect of the invention provides a method of determining thepresence in a sample of antibodies specifically reactive with atransferrin receptor protein of a strain of Moraxella having a molecularmass of about 80 to about 90 kDa, comprising the steps of:

(a) contacting the sample with the transferrin receptor protein asprovided herein to produce complexes comprising the transferrin receptorprotein and any said antibodies present in the sample specificallyreactive therewith; and

(b) determining production of the complexes.

In a further aspect of the invention, there is provided a method ofdetermining the presence in a sample of a transferrin receptor proteinof a strain of Moraxella having a molecular mass of about 80 to about 90kDa, in a sample comprising the steps of:

(a) immunizing a subject with the immunogenic composition as providedherein, to produce antibodies specific for the transferrin receptorprotein;

(b) contacting the sample with the antibodies to produce complexescomprising any outer membrane protein present in the sample and saidouter membrane protein specific antibodies; and

(c) determining production of the complexes.

The transferrin receptor protein may be part of a Moraxella catarrhalisstrain.

A further aspect of the invention provides a diagnostic kit fordetermining the presence of antibodies in a sample specifically reactivewith the transferrin receptor protein of a strain of Moraxella having amolecular mass of about 80 to about 90 kDa, comprising:

(a) the transferrin receptor protein as provided herein;

(b) means for contacting the transferrin receptor protein with thesample to produce complexes comprising the transferrin receptor proteinand any said antibodies present in the sample; and

(c) means for determining production of the complexes,

The invention also provides a diagnostic kit for detecting the presence,in a sample, of a transferrin receptor protein of a strain of Moraxellahaving a molecular mass of about 80 to about 90 kDa comprising:

(a) an antibody specific for the about 80 to about 90 kDa transferrinreceptor protein as provided herein;

(b) means for contacting the antibody with the sample to produce acomplex comprising the transferrin receptor protein and transferrinreceptor protein-specific antibody; and

(c) means for determining production of the complex.

The present invention provides, in a further aspect, a method ofproducing a vaccine, comprising administering the immunogeniccomposition provided herein to a test host to determine an amount and afrequency of administration thereof to confer protection against diseasecaused by a Moraxella strain that produces a transferrin receptorprotein having an apparent molecular mass of about 80 to about 90 kDa,as determined by SDS-PAGE or a protein capable of inducing antibodies ina test host specifically reactive with the transferrin protein; andformulating the immunogenic composition in a form suitable foradministration to a treated host, including a human host, in accordancewith the determined amount and frequency of administration.

In an additional aspect of the invention, there is provided a method ofproducing monoclonal antibodies specific for a transferrin receptorprotein of a Moraxella strain having an apparent molecular mass of about80 to about 90 kDa, comprising:

(a) administrating the transferrin receptor protein provided herein toat least one mouse to produce at least one immunized mouse,

(b) removing B-lymphocytes from the at least one immunized mouse;

(c) fusing the B-lymphocytes from the at least one immunized mouse withmyeloma cells, thereby producing hybridomas;

(d) cloning the hybridomas;

(e) selecting clones which produce anti-transferrin receptor proteinantibody;

(f) culturing the anti-transferrin receptor protein antibody-producingclones; and then

(g) isolating anti-transferrin receptor protein antibodies from thecultures.

In a further aspect of the invention, there is provided a method ofproducing an isolated and purified non-denatured transferrin receptorprotein of a strain of Moraxella, such as M. catarrhalis having amolecular mass of about 80 to about 90 kDa, comprising the steps of:

(a) providing a cell mass of the Moraxella strain;

(b) selectively extracting aqueous soluble proteins from the cell massto provide a first supernatant and a first pellet;

(c) separating the first supernatant from the first pellet;

(d) selectively solubilizing at least a transferrin receptor proteinhaving a molecular mass of about 80 to 90 kDa from the first pellet toprovide a second supernatant and a second pellet;

(e) separating the second supernatant from the second pellet; and

(f) purifying transferrin receptor protein having a molecular mass ofabout 80 to about 90 kDa in the second supernatant substantially freefrom other Moraxella proteins solubilized from the first pellet in theselective solubilization step.

The aqueous soluble proteins may be selectively extracted from the cellmass by contacting the cell mass with a buffered aqueous solution, whichmay comprise about 50 mM to about 1 M Tris-HCl at a pH of about 7 toabout 8.5, sonication of the cell mass to disrupt the same, andcentrifugation to form the first pellet and the first supernatant. Theselective solubilization step may be effected by contacting the firstpellet at least once with a buffered aqueous solution, which may beabout 20 mM to about 1 M Tris-HCl, comprising a detergent, which may beabout 0.2 to about 2 wt. % of TRITON X-100 (Trademark for non-ionicdetergent which is octadecylphenol (ethylene glycol) 10), and asolubilizing agent, which may be about 2 to about 20 mM of EDTA,sonication of the first pellet to disrupt the same and centrifugation toform the second pellet and the second supernatant. Such contacting,sonication and centrifugation steps may be effected at least twice andsupernatant from each such step is then pooled to provide the secondsupernatant.

The purification step may be effected by multiple chromatographic columnoperations, which may include:

(a) a first chromatographic operation on a first chromatography column,which may be a DEAE-Sephacel column through which the transferrinreceptor selectively flows and on which contaminating proteins bind,such as by use of a buffer comprising about 10 mM to about 1 M Tris-HClat a pH of about 7 to about 8.5,

(b) a second chromatographic operation on a second chromatography columncomprising a cation-exchange matrix, such as SE-Cellulose/D529, throughwhich the transferrin receptor selectively flows and on whichcontaminating proteins bind, such as by use of a buffer comprising about10 mM to about 1 M Tris-HCl at a pH of about 7 to about 8.5,

(c) a third chromatographic operation on a third chromatography column,such as hydroxyapatite, on 5 which the transferrin receptor protein isselectively bound in preference to OMP B2 protein, such as by loadingthe column at a pH of about 7 to about 8.5, and

(d) eluting the transferrin receptor protein from 10 the thirdchromatography column, such as by using a buffer solution having a pH ofabout 7 to about 8.5 containing about 100 to about 250 mM KH₂PO₄.

The preparation procedure provided herein results in the isolation ofthe transferrin receptor protein in a purified form and in anondenatured form.

In this application, the term “a transferrin receptor protein (Tfr2) ofa Moraxella strain having an apparent molecular mass of about 80 toabout 90 kDa” is used to define a family of transferrin receptorproteins of M. catarrhalis having molecular mass of between about 80 andabout 90 kDa and includes proteins having variations in their amino acidsequences including those naturally occurring in various strains ofMoraxella. In this application, a first protein is a “functional analog”of a second protein if the first protein is immunologically related toand/or has the same function as the second protein. The functionalanalog may be, for example, a fragment of the protein or a substitution,addition or deletion mutant thereof.

Advantages of the present invention include:

a method for isolating purified transferrin receptor protein having anapparent molecular mass of about 80 to about 90 kDa from a Moraxellastrain that produces the transferrin receptor protein, includingMoraxella catarrhalis;

an isolated and purified non-denatured transferrin receptor proteinhaving an apparent molecular mass of about 80 to about 90 kDa andisolatable from a Moraxella strain; and

diagnostic kits and immunological reagents for specific identificationof Moraxella and hosts infected thereby.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be further understood from the followingdescription with reference to the FIGURES, in which:

FIGS. 1A, 1B, 1C and 1D are a flow diagram of a method for purifyingtransferrin receptor protein from Moraxella catarrhalis, according toone embodiment of the invention;

FIG. 2 shows the ability of purified transferrin receptor to bind humantransferrin;

FIGS. 3A and 3B shows an analysis by SDS-PAGE of isolated and purifiedtransferrin receptor protein having an apparent molecular mass of about80 to about 90 kDa;

FIGS. 4A, 4B and 4C show an immunoblot analysis of purified transferrinreceptor protein; and

FIGS. 5A and 5B are immunoblots to demonstrate the ability of guinea piganti-Tfr antisera produced by immunization with isolated and purifiedtransferrin receptor protein to specifically distinguish between M.catarrhalis and other bacterial pathogens that cause otitis media.

GENERAL DESCRIPTION OF INVENTION

The present invention provides novel techniques which can be employedfor preparing purified transferrin receptor protein Tfr1 from M.Catarrhalis. Any M. Catarrhalis strain that produces Tfr2 may beconveniently used to provide the isolated and purified Tfr2 as providedherein. Such strains are generally available from clinical sources andfrom bacterial culture collections. Appropriate strains of M.catarrhalis may include M. catarrhalis 5191, 135 and 4223.

Referring to FIG. 1, there is illustrated a flow diagram of a method forpurifying a transferrin protein from M. catarrhalis having an apparentmolecular mass of about 80 to about 90 kDa, as determined by SDS-PAGEaccording to one embodiment of the invention. The various operationsdescribed below may be effected at ambient temperatures, generally about4° to about 30° C. Whole cells are contacted with an aqueous buffermedium, which may contain about 10 mM to about 1 M Tris-HCl at pH about7 to about 8.5 and disrupted by sonication to produce, followingcentrifugation, a first supernatant (S1) and a first pellet (PPT1). Thesupernatant (S1) contains a substantial proportion of the solubleprotein from the cell pellet and is discarded. The remaining pellet(PPT1) is selectively detergent extracted to remove any residual solubleproteins and to solubilize membrane proteins, including the TfR2protein, from the pellet. Such selective extraction may be effected inany convenient manner. One such procedure may involve multiple detergentextractions of the pellet. In particular, a first detergent extractionmay be effected using Triton X-100 and EDTA followed by centrifugationto form a second pellet (PPT2) and a second supernatant (S2), which isretained. Following separation of the second supernatant (S2), theresidual pellet PPT2 is again detergent extracted using Triton X-100 andEDTA, followed by centrifugation. The resulting third supernatant (S3),following separation from the residual third pellet (PPT3), which isdiscarded, is combined with the second supernatant (S2) from the firstextraction to provide pooled supernatants (TfR2-1). Such Triton X-100extractions may be effected using a solution of concentration about 0.2to about 2 wt % Triton X-100 and about 2 to about 20 mM EDTA underbuffer conditions, such as a pH about 7 to about 8.5, using about 10 mMto about 1 M Tris.

The pooled supernatants (TfR2-1) contain the TfR2 rotein, as well ascontaminating M. catarrhalis proteins, including the OMP B2 proteinwhich has the same approximate molecular mass (see Lanes 3 and 5, FIG.2). The pooled supernatants (TfR2-1) are processed to remove suchimpurities in a series of column chromatography operations.

In a first such column chromatography operation, the pooled supernatants(TfR2-1) may be applied to a DEAE-Sephacel column or other suitablecolumn, suitably buffered, such as with about 10 mM to about 1 MTris-HCl at a pH of about 7 to about 8.5, to permit the TfR2 protein topass through the column, while impurities are bound to the column. TheDEAE-Sephacel column may be washed before further processing of the flowthrough.

The flow through and wash from the DEAE-Sephacel column (TfR2-2) thenmay be applied to a cation-exchange column, for example,SE-cellulose/D529 or S-Sepharose, suitably buffered, such as with about10 mM to about 1 M Tris-HCl at pH about 7 to about 8.5, to permit TfR2protein to pass through the column, while impurities are bound to thecolumns. The cation-exchange column may be washed before furtherprocessing of the flow-through (see lane 4, FIG. 2).

The flow-through and wash from the cation-exchange column (TfR2-3),which still contains the OMP B2 protein, as well as small quantities ofresidual proteinaceous contaminants, then may be applied to ahydroxyapatite column under such buffer conditions that the TfR2 proteinbinds to the column in preference to the OMP B2 protein and othercontaminating proteins, which pass through the column. Such conditionsmay be provided using about 5 to about 50 mM potassium phosphate bufferat pH about 6 to about 8.5. The flow-through contains the contaminatingproteins (see lane 5, FIG. 2).

Following repeated washing with the buffer to remove column-retainedcontaminants, the TfR2 protein then is eluted from the hydroxyapatitecolumn using a suitable buffer, such as about 150 to about 250 mMpotassium phosphate buffer solution at pH about 7 to about 8.5. Theeluted fractions are collected, aliquots analyzed by SDS-PAGE andTfR2-containing fractions pooled. The pooled fractions may beconcentrated to provide a TfR2 protein-containing solution (see lane 6,FIG. 2). By this procedure, there is provided an aqueous solution of anisolated and purified non-denatured TfR2 protein. The aqueous TfR2solution may be provided in any convenient concentration consistent withthe intended use of the material, generally about 100 to about 200μg/ml.

Referring to FIG. 2, there is shown an analysis of the purity oftransferrin receptor protein by SDS-PAGE analysis, purified by themethod described herein as typified by that schematically shown in FIG.1. In FIG. 2, Lane 1 shows the M. catarrhalis cell lysate. Lane 2 showsthe pooled supernatants (TfR2-1). Lane 3 shows the run-through fractionfollowing DEAE-Sephacel column chromatography (TfR2-2) and Lane 4 showsthe run-through fraction of SE-cellulose/D529 column chromatography(TfR2-3). Lane 5 shows the flow-through fraction of HTP and contains theOMP B2 protein. Lane 6 shows the HTP bound fraction containing TfR2.Lane 7 shows transferrin receptor protein TfR2 isolated by affinitychromatography essentially as described in ref. 29. The method forpurification of TfR2 protein as provided herein produces at least a 70%pure TfR2 protein preparation. The illustrated preparation in Lane 6 inFIG. 2 is at least 95% pure, as determined by densitometry scanning.

Referring to FIG. 3, there is illustrated the ability of purified TfR2of M. catarrhalis as provided herein to specifically bind humantransferrin. Proteins were separated by SDS-PAGE and in vitrotransferrin binding assessed essentially as described in ref. 27. Lane 1contains TfR2 of M. catarrhalis isolated by affinity chromatographyessentially as described in ref. 29. Lane 2 contains TfR2 from M.catarrhalis purified as described herein. Lane 3 contains M. catarrhalisOMPB2 protein. As can be seen from FIG. 3, the transferrin bindingprotein preparations specifically bind transferrin. These resultsconfirm the isolation of TfR2 from M. catarrhalis by the method providedherein.

To be useful as a component of immunogenic compositions (includingvaccines) and as an antigen in diagnostic embodiments, the transferrinreceptor protein purified as described herein should advantageously becapable of generating antibodies that recognize or neutralize M.catarrhalis strains, including a plurality thereof.

Referring to FIGS. 4(a), 4(b) and 4(c), there is shown an immunoblotanalysis of TfR2 as provided herein and shows the specific separation ofTfR2 from OMP B2 protein. In each of the FIGURES, sample 1 contains OMPB2 protein and sample 2 contains TfR2 protein. The M. catarrhalisstrains were a: strain 5191; b: strain 135; and c: strain 4223. Sampleswere separated by SDS-PAGE. Sample 3 was purified TfR2. FIG. 4(a) showsa Coomassie blue stained gel. FIG. 4(b) shows an immunoblot using ananti-TfR2 antiserum. FIG. 4(c) shows an immunoblot using an anti-OMP B2antiserum. The results of FIG. 4 confirm the specific purification oftransferrin receptor protein TfR2 from M. catarrhalis by the methodprovided herein and particularly substantially free from OMBP2 protein.

Referring to FIG. 5(b), there is illustrated an immunoblot showing theability of guinea pigs anti-TfR antisera produced by immunizing guineapigs with purified TfR protein as provided herein to recognize TfR2protein from M. catarrhalis isolated from a variety of sources. Thesamples tested were as follows:

Lane M. catarrhalis Source 1 Purified TfR2 M. catarrhalis 4223 2  56Middle ear fluid 3 4223 Middle ear fluid

In one embodiment of the present invention, the isolated and purifiedTfR2 protein as provided herein is useful for generating antibodies thatcan be used to specifically distinguish M. catarrhalis from otherbacterial pathogens that cause otitis media. Thus referring to FIGS.5(a) and 5(b), there is illustrated an SDS-PAGE and an immunoblotrespectively showing the specific reactivity of guinea pig anti-TfR2antisera produced by immunizing guinea pigs with TfR2 protein asprovided herein. The samples analyzed were as follows:

Lane Bacterium Source 1 TfR2 protein M. catarrhalis 4223 2 M.catarrhalis 56 Middle ear fluid 3 M. catarrhalis 4223 Middle ear fluid 4H. influenzae strain 12 5 H. influenzae strain 30 Otitis media isolate 6H. influenzae LCOC 1 Otitis media isolate 7 S. pneumoniae ATCC 6303 8 S.pneumoniae ATCC 6304 9 S. pneumoniae ATCC 6314

The results shown in FIG. 5(b) clearly show the usefulness ofTfR2-specific antisera as provided herein to distinguish betweenbacterial pathogens that produce diseases with similar clinicalsymptoms.

Results shown in Table 1 below illustrate the ability of guinea piganti-TfR2 antisera produced by immunization with TfR2 protein asprovided herein, to kill M. catarrhalis. The results show that antiseraproduced by immunization with TfR2 protein isolated from strain 4223were bactericidal against a homologous non-clumping M. catarrhalisstrain RH408 derived from strain 4223. The ability of the isolated andpurified TfR2 protein as provided herein to generate bactericidalantibodies is in vivo evidence of utility of the TfR2 protein asprovided herein as a vaccine to protect against diseases caused by M.catarrhalis.

Thus, in accordance with another aspect of the present invention, thereis provided a vaccine against Moraxella or other bacterial pathogensthat produce TfR2 protein or produce a protein capable of inducingantibodies that specifically recognize TfR2, comprising animmunogenically-effective amount of TfR2 protein as provided herein anda physiologically-acceptable carrier therefor. The TfR2 protein providedherein also may be used as a carrier protein for hapten, polysaccharidesor peptides to make a conjugate vaccine against antigenic determinantsunrelated to TfR2.

The TfR2 protein provided herein is useful as a diagnostic reagent, asan antigen or for the generation of anti-TfR2 antibodies, antigen forvaccination against the diseases caused by species of Moraxella.

In additional embodiments of the present invention, the TfR2 protein asprovided herein may be used as a carrier molecule to prepare chimericmolecules and conjugate vaccines (including glycoconjugates) againstpathogenic bacteria, including encapsulated bacteria. Thus, for example,glycoconjugates of the present invention may be used to conferprotection against disease and infection caused by any bacteria havingpolysaccharide antigens including lipooligosaccharides (LOS) and PRP.Such bacterial pathogens may include, for example, Haemophilusinfluenzae, Streptococcus pneumoniae, Escherichia coli, Neisseriameningitidis, Salmonella typhi, Streptococcus mutants, Cryptococcusneoformans, Klebsiella, Staphylococcus aureus and Pseudomonasaeruginosa. Particular antigens which can be conjugated to TfR2 proteinand methods to achieve such conjugations are described in published PCTapplication WO 94/12641, assigned to the assignee hereof and thedisclosure of which is hereby incorporated by reference thereto.

In another embodiment, the carrier function of TfR2 protein may be used,for example, to induce immunity toward abnormal polysaccharides of tumorcells, or to produce anti-tumor antibodies that can be conjugated tochemotherapeutic or bioactive agents.

The present invention extends to the TfR2 protein for use as apharmaceutical substance, particularly as an active ingredient in avaccine against diseases caused by infection of a strain of Moraxella.

In a further aspect, the invention provides the use of the TfR2 proteinfor the preparation of a medicament for immunization against diseasescaused by infection with a strain of Moraxella.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of Moraxella infections andthe generation of immunological reagents. A further non-limitingdiscussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from immunogenic transferrin receptor, proteins or analogs asdisclosed herein. Preferably, the antigenic material is extensivelydialyzed to remove undesired small molecular weight molecules and/ormyophilized for more ready formulation into a desired vehicle. Theimmunogenic composition elicits an immune response which producesantibodies, including anti-transferrin receptor antibodies andantibodies that are opsonizing or bactericidal. Should the vaccinatedsubject be challenged by Moraxella or other bacteria that produce atransferrin receptor, the antibodies bind to the transferrin receptorand thereby prevent access of the bacteria to an iron source which isrequired for viability. Furthermore, opsonizing or bactericidal anti-TfRantibodies may also provide protection by alternative mechanisms.

Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The transferrin receptorprotein may be mixed with pharmaceutically acceptable excipients whichare compatible therewith. Such excipients may include, water, saline,dextrose, glycerol, ethanol, and combinations thereof. The immunogeniccompositions and vaccines may further contain auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness thereof. Immunogenic compositions and vaccinesmay be administered parenterally, by injection subcutaneously orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. Alternatively, othermodes of administration including suppositories and oral formulationsmay be desirable. For suppositories, binders and carriers may include,for example, polyalkalene glycols or triglycerides. Such suppositoriesmay be formed from mixtures containing the active ingredient(s) in therange of about 0.5 to about 10%, preferably about 1 to 2%. Oralformulations may include normally employed incipients such as, forexample, pharmaceutical grades of saccharine, cellulose and magnesiumcarbonate. These compositions can take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain about 1 to 95% of the transferrin receptor protein,preferably about 20 to about 75%.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies, the degree of protection desired and if needed, to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of micrograms of the transferrinreceptor protein per vaccination. Suitable regimes for initialadministration and booster doses are also variable, but may include aninitial administration followed by subsequent administrations. Thedosage may also depend on the route of administration and will varyaccording to the size of the host.

The concentration of the transferrin receptor protein in an immunogeniccomposition according to the invention is in general about 1 to 95%. Avaccine which contains antigenic material of only one pathogen is amonovalent vaccine. Vaccines which contain antigenic material of severalpathogens are combined vaccines and also belong to the presentinvention. Such combined vaccines contain, for example, material fromvarious pathogens or from various strains of the same pathogen, or fromcombinations of various pathogens.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune responses. Thus,adjuvants have been identified that enhance the immune response toantigens delivered parenterally. Some of these adjuvants are toxic,however, and can cause undesirable side-effects, making them unsuitablefor use in humans and many animals. Indeed, only aluminum hydroxide andaluminum phosphate (collectively commonly referred to as alum) areroutinely used as adjuvants in human and veterinary vaccines. Theefficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum. While the usefulness of alum is well establishedfor some applications, it has limitations. For example, alum isineffective for influenza vaccination and inconsistently elicits a cellmediated immune response. The antibodies elicited by alum-adjuvantedantigens are mainly of the IgG1 isotype in the mouse, which may not beoptimal for protection by some vaccinal agents.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are often emulsified in adjuvants. Manyadjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant, FCA), cytolysis (saponins andPluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

Desirable characteristics of ideal adjuvants include:

(1) lack of toxicity;

(2) ability to stimulate a long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) ability to elicit both CMI and HIR to antigens administered byvarious routes, if required;

(5) synergy with other adjuvants;

(6) capability of selectively interacting with populations of antigenpresenting cells (APC);

(7) ability to specifically elicit appropriate T_(H)1 or T_(H)2cell-specific immune responses; and

(8) ability to selectively increase appropriate antibody isotype levels(for example, IgA) against antigens.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989 whichis incorporated herein by reference thereto teaches glycolipid analoguesincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates,each of which is substituted in the sugar residue by an amino acid, asimmuno-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No.4,855,283 and ref. 33) reported that N-glycolipid analogs displayingstructural similarities to the naturally-occurring glycolipids, such asglycosphingolipids and glycoglycerolipids, are capable of elicitingstrong immune responses in both herpes simplex virus vaccine andpseudorabies virus vaccine. Some glycolipids have been synthesized fromlong chain-alkylamines and fatty acids that are linked directly with thesugars through the anomeric carbon atom, to mimic the functions of thenaturally occurring lipid residues.

U.S. Pat. No. 4,258,029 granted to Moloney, assigned to the assigneehereof and incorporated herein by reference thereto, teaches thatoctadecyl tyrosine hydrochloride (OTH) functioned as an adjuvant whencomplexed with tetanus toxoid and formalin inactivated type I, II andIII poliomyelitis virus vaccine. Also, Nixon-George et al. (ref. 34),reported that octadecyl esters of aromatic amino acids complexed with arecombinant hepatitis B surface antigen, enhanced the host immuneresponses against hepatitis B virus.

2. Immunoassays

The transferrin receptor protein of the present invention is useful asan immunogen for the generation of anti-transferrin receptor proteinantibodies, as an antigen in immunoassays including enzyme-linkedimmunosorbent assays (ELISA), RIAs and other non-enzyme linked antibodybinding assays or procedures known in the art for the detection ofanti-bacterial, anti-Moraxella, and anti-TfR antibodies. In ELISAassays, the transferrin receptor protein is immobilized onto a selectedsurface, for example, a surface capable of binding proteins such as thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed transferrin receptor protein, a nonspecificprotein such as a solution of bovine serum albumin (BSA) that is knownto be antigenically neutral with regard to the test sample may be boundto the selected surface. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from 2 to 4 hours, at temperatures suchas of the order of about 25 to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween or a borate buffer. Following formation of specificimmunocomplexes between the test sample and the bound transferrinreceptor protein, and subsequent washing, the occurrence, and evenamount, of immunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and in generalIgG. To provide detecting means, the second antibody may have anassociated activity such as an enzymatic activity that will generate,for example, a colour development upon incubating with an appropriatechromogenic substrate. Quantification may then be achieved by measuringthe degree of colour generation using, for example, a spectrophotometer.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example illustrates the growth of M. catarrhalis.

The M. catarrhalis strains used were 135, 4223 (ref. 35), 5191 (ref. 35)(all middle ear isolates), ATCC 25240, Q8 (expectorate isolate) andRH408.

To provide cells for isolation of TfR2, M. catarrhalis strains wereroutinely maintained on chocolate agar plates (BBL).

M. catarrhalis strain 4223 was inoculated into 20 mL of brain heartinfusion (BHI) broth. The culture was incubated overnight with aerationat 37° C. For growth under iron-restricted conditions, one mL of theovernight-culture was inoculated into 20 mL of BHI broth containing 25μM EDDA and the culture was grown at 37° C. for approximately 3 to 4 h.Cells grown to mid-log phase (A₅₇₈>0.5) were harvested by centrifugationat 10,000×g for 20 min. The pellet was used for extraction oftransferrin receptor (TfR2) protein as described in Example 3 below.

Example 2

This Example illustrates the generation of a non-clumping strain (RH408)of M. catarrhalis.

M. catarrhalis strain 4223 was inoculated into several flasks containing20 mL of BHI broth, and the cultures were incubated with shaking (170rpm) overnight at 37° C. Five mL of each overnight culture weretransferred to individual 1 mL tubes, and were left sitting undisturbedat room temperature for 3 to 8 hours, to allow bacteria to sediment. Onehundred μL of the cleared upper phase of each culture medium were usedto inoculate 25 mL of BHI broth and cultures were incubated overnight at37° C., as described above. This passaging was repeated six times, using25 μL of cleared medium to inoculate 25 mL of BHI for each overnightculture. Non-clumping bacterial cultures were identified by measuringthe A₅₇₈ at intervals over a 3 hour time period, in order to compare thesedimentation rates of the passaged strains to that of the original M.catarrhalis strain 4223 culture. Non-clumping mutants, including M.catarrhalis RH408, did not aggregate during the three hour time period.On BHI agar plates, strain RH408 had a colony morphology typical for allnon-clumping strains. Strain RH408 is deposited at the American TypeCulture Collection (ATCC) at 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., under the terms of the Budapest Treaty, on Dec. 13,1994 and was given ATCC Accession No. 55637.

Example 3

This Example illustrates the extraction of transferrin receptor proteinTfR2 by affinity purification.

Affinity purified TfR2 was prepared from total membranes as described inreference 29 using Sepharose 4B (Pharmacia) conjugated human transferrin(Sigma). Bacterial receptor proteins bound to the affinity matrix wereselectively eluted using a buffer of 50 mM Tris-HCl pH 8.0, 1M NaCl, 10mM EDTA, 0.05% sodium lauryl sarkosinate, 2M guanidine. Eluted proteinswere dialized against 50 mM Tris-HCl pH 8.0.

Example 4

This Example illustrates the extraction and purification of transferrinreceptor protein TfR2.

Transferrin receptor protein was isolated from M. catarrhalis by theprocedure generally illustrated in FIG. 1.

A cell pellet from iron-deprived overnight cultures of M. catarrhaliswas resuspended in 50 mM Tris-HCl, pH 8.0, and disrupted by sonication.The sonicate was centrifuged at 20,000×g for 30 minutes and theresultant supernatant (S1), which contained soluble proteins, wasdiscarded. The pellet (PPT1) was suspended in 50 mM Tris, pH 8.0containing 0.5% Triton X-100 and 10 mM EDTA. This extraction stepsolubilized TfR2. The suspension was centrifuged at 20,000×g for 20minutes to remove insoluble material and the supernatant was applied toa DEAE-Sephacel (Pharmacia) column equilibrated in 50 mM Tris-HClbuffer, pH 8.0. TfR2 was recovered in the flow-through fraction and wasfurther purified using a SE-cellulose D529 column equilibrated in 50 mMTris-HCl, pH 8.0. The flow-through fraction containing TfR2 was nextloaded onto a hydroxyapatite (HTP) column equilibrated in 10 mMphosphate buffer, pH 8.0. This step separated OMP B2 (present in the HTPflow-through fraction) from TfR2 which remained bound to the HTP. Afterextensive washing of HTP column with 50 mM phosphate buffer, pH 8.0,TfR2 was eluted from the matrix with a 200 mM phosphate buffer, pH 8.0.The purity of TfR2 was assessed by SDS-PAGE analysis.

Example 5

This Example describes the purification of M. catarrhalis outer membraneprotein B2 OMP B2.

The flow-through fraction of HTP column was analyzed by 12.5% SDS-PAGE.Following electrophoresis, a gel slice corresponding to the OMP B2protein band was cut from the gel. Protein was recovered from the gelslice by electroelution for 12 hours at 100 volts in elution buffer(15mM NH₄CO₃/0.1% SDS).

Example 6

This Example illustrates the immunization of guinea pigs.

Guinea pigs (Charles River, Quebec) were immunized intramuscularly(i.m.) on day 1 with a 5 μg dose of either TfR2 or OMP B2 proteinemulsified in complete Freund's adjuvant (CFA). Animals were boosted ondays +14 and +28 with the same dose of protein emulsified in incompleteFreund's adjuvant (IFA). Blood samples were taken on day +42.

Example 7

This Example describes the analysis of purified TfR2 and OMP B2proteins.

Proteins were separated by SDS-PAGE as described by Lugtenberg et al(ref. 36) using 11.5% or 12.5% (w/v) acrylamide (BRL) in the separatinggels. Proteins were visualized by staining gels with Coomassie brilliantblue (BioRad). Protein standard molecular mass markers were from BioRadand Pharmacia.

Proteins separated by SDS-PAGE were electroblotted by the method ofTowbin et al (ref. 37) onto polyvinylidene difluoride membranes (PVDF,Millipore). For immunoblots, antisera were used at dilutions of 1:1,000.Recombinant protein G horseradish peroxidase conjugate (Zymed) was usedas a reporter at a dilution of 1:4,000. Blots were developed usingeither a olourimetric reaction (4CN/DAB; Pierce) or a chemiluminescentreaction (LumiGlo; Kirkegaard and Perry). Procedures recommended by themanufacturers were followed for both detection methods. Prestainedmolecular mass markers were purchased from BioRad.

In vitro transferrin-binding activity of the purified proteins wasassessed following the method described by Schryvers and Lee (ref. 27)with some modifications. Briefly, protein samples were separated bySDS-PAGE and then electrophoretically transferred to PCDF membranes. Themembranes were incubated with horseradish peroxidase conjugated humantransferrin (1:50 dilution; Jackson ImmunoResearch) overnight at 4° C.Blots were developed using the LumiGlo chemiluminescent reactiondescribed above.

Example 8

This Example illustrates the bactericidal assay against B. catarrhalis.

Samples (25 μL) of antiserum, were heated to 56° C. for 30 minutes toremove complement activity and diluted 1:8 in veronal buffer (NaCl 8.0g/L, NaHCO₃ 0.25 g/L, Na Barbiturate 0.30 g/L, Barbituric acid 0.45 g/L,MgCl₂. 6H₂O 0.1 g/L, CaCl₂.₂H₂O 0.05 g/L) containing 0.1% BSA (VBS), andthen added to the first well of a 96-well Nunc microtiter plate.Two-fold serial dilutions of antisera in VBS were placed in theremaining wells. Bacterial cells grown to an OD₅₇₈>0.5 were diluted1:200,000 in VBS and 25-μL portions of the bacterial suspension wereadded to each well. A guinea pig complement (Biowhittaker, Walkersville,Md.) was diluted 1:10 in VBS and 25 μL of the solution were added toeach well to initiate reactions. The plates were incubated at 37° C. for60 min, and 50 μL of each reaction mixture was then plated ontoMueller-Hinton agar plate containing 2.2% Mueller-Hinton broth and 1.5%agar. After incubation at 37° C. for 48 hours, colonies were counted todetermine the bactericidal titer (the reciprocal of the highest dilutionof antiserum capable of killing greater than 50% of bacteria comparedwith controls containing pre-immune sera).

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides atransferrin receptor protein having an apparent molecular mass of about80 kDa to about 90 kDa and isolated from M. catarrhalis, methods ofmaking the same and uses thereof as immunogenic compositions anddiagnostic embodiments. Modifications are possible within the scope ofthis invention.

TABLE 1 Bactericidal activity of guinea pig antisera obtained byimmunization with transferrin receptor protein (TfR2) from M.catarrhalis 4223 Titre¹ Immunogen Pre-immune Post-immune TfR2 <3.0 13.5OMP B2 <3.0 <3.0 ¹Bactericidal titres: expressed in log₂ as the dilutionof antiserum capable of killing 50% of cells of M. catarrhalis 4223.

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What we claim is:
 1. An isolated and purified non-denatured transferrinreceptor protein of a strain of Moraxella catarrhalis having an apparentmolecular mass of about 80 to about 90 kDa, as determined by sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
 2. Theprotein of claim 1 wherein the strain is Moraxella catarrhalis 4223,5191 or
 135. 3. The protein of claim 1 which is at least about 70 wt %pure.
 4. The protein of claim 3 which is at least about 90 wt % pure. 5.A composition, comprising an aqueous solution of an isolated andpurified non-denatured transferrin receptor protein of a Moraxellacatarrhalis strain having an apparent molecular mass of about 80 toabout 90 kDa, as determined by sodium dodcecyl sulphate polacrylamidegel electrophoresis (SDS-PAGE).
 6. The protein of claim 1 free from theOMP B2 protein of the Moraxella catarrhalis strain.
 7. The protein ofclaim 1 free from the transferrin receptor protein of the Moraxellacatarrhalis strain having an apparent molecular mass of about 105 kDa.